Acid-Base Properties of Amino Acids - Online Tutor, Practice Problems & Exam Prep

Amino acids exist as zwitterions at physiological pH (around 7.4), featuring both positive and negative charges that balance to a net charge of zero. Their amphoteric nature allows them to act as acids or bases depending on the pH, influenced by their pKa values. For instance, at pH levels below the pKa of the carboxylic acid (approximately 2), amino acids become protonated, while at pH levels above the pKa of the amine (around 9), they lose protons. Additionally, seven amino acids possess ionizable side chains, requiring consideration of their pKa values for accurate charge determination.

Why Amino Acids Exist as Zwitterions

Up to this point, we've been representing amino acids as these neutral structures. But it turns out that at physiological pH, meaning the pH inside your body, these amino acids don't usually look neutral. They're usually charged. So how are we going to know what types of charges to place on them? Well, that's what we're going to focus on in this video when we talk about the acid and base properties of amino acids. So at physiological pH, which is around, you know, people say 7, but it's actually like 7.4. If you're at pH 7, you're going to be in the hospital. That's not good. You need to be a little bit basic. 7.4 is the body's pH. And at that pH, amino acids exist in this state called zwitterions. You've probably heard the word zwitterion before, maybe not. But what it means is just it's a net neutral molecule with separation of charges. So let's write that in, separation of charges. So that means that, overall, the net charge is 0 but there's a positive and a negative or multiple and multiple negatives that all cancel out. Okay? So why does it exist like that? Because it's counterintuitive. Like this is the actual equilibrium, what it looks like. The amino acid starts off the way that we drew it, that we've been drawing it this whole time. It has a regular carboxylic acid. It has a regular amine. And then at physiological pH, the equilibrium shifts so that you get an anion on the O and a cation on the positive. If you think about it from a chemistry perspective, that doesn't make a whole lot of sense at first because you're always taught to think that the neutral structure is the most stable. So wouldn't it be more stable for it to stay over in that side than for it to spontaneously turn into this? Why would charges be a good thing? Why would it want to look like this? Well, the explanation goes back all the way back to our acid-base chemistry in inorganic chemistry. In a topic called determining acid-base equilibrium, we learned that the side of an equilibrium that is favored is the one with the highest pKa. We learned if you have an acid-base reaction, the side with the acid with the highest pKa is going to be the weakest acid and that's going to be the one that's the most favored. Well, that's what's happening here because it turns out that all that's happening is that this hydrogen, which I'm highlighting in red and I'm also going to highlight in yellow, is moving locations. It's getting off of the O and instead, it's joining this nitrogen as being one of the 3 Hs on that nitrogen. So it's basically moving over, okay? If you were to think of it as a mechanism, the N is actually grabbing the H and giving electrons to the O. Does that kind of make sense? Well, why would that make sense? Well because that hydrogen has to choose which acid it wants to be on. Does it want to be on a carboxylic acid, which is a pretty moderately decently strong acid? Or does it want to be on ammonium, which isn't as strong of an acid? Going back to organic chemistry, do you remember the pKa that we memorized for carboxylic acids? It was around 5, okay? And it turns out that amino acids are a little bit more acidic because they have extra groups around like nitrogen that stabilize the anion. So actually, the pKa of most carboxylic acids in amino acids is around 2. So let's write that in. The pKa is roughly 2 of that hydrogen, okay? Once that hydrogen moves over and joins the nitrogen, we learned in organic chemistry 1 that a nitrogen with a positive charge tends to have a pKa of around 10, okay? And actually, it turns out that we are very close. It's usually around 9 for amino acids. So, basically, the hydrogen has a choice. Does it stay on the acid that has a pKa of 2, or does it move to the acid with a pKa of 9? And the answer is clear from our acid-base equilibrium video, you pick the side with the highest pKa. So that's why the hydrogen doesn't want to be over here. It prefers to be on the acid with the highest pKa because that's going to be the one that's the most favored. It's the weakest acid. So that is why that's the chemistry explanation for why amino acids exist in zwitterions. Okay? So how are we going to know what types of zwitterions to draw? And it turns out that differences in the pH will actually change the charges. So how are we going to do that? We're going to find out in the next video when we talk about predominant forms.

Determining Predominant Forms

Like water, amino acids are amphoteric, and "amphoteric" is just a fancy word for a molecule that can react either as an acid or as a base depending on the situation. And the reason I mentioned water is because water is the most famous example of an amphoteric molecule. Remember that water could either accept a proton or it could donate a proton based on its surroundings, based on the pKa and the pH of the solution. Well, the same thing is true of amino acids because amino acids have an acidic carboxylic acid group and a basic amine group. So, we're going to need to figure out when it's reacting as an acid and when it's reacting as a base. And for that, we're going to be using exact pKa values. We use exact pKa values given to us on a table to figure out at a certain pH, does that amino acid exist in a charged state or is it in a neutral state, positive or negative. Now, I've gone ahead and provided all these pKas for you in your amino acid breakdown sheet. We're not going to look at it right now, but I just want to remind you, remember all those numbers you saw next to the amino acids? This is what we're using them for. We're going to use them to determine the charges of the amino acids at different pHs, okay?

Before we look at an example, I want to remind you about the theory behind this. Remember that in general chemistry, there was an equation that we learned called the Henderson-Hasselbalch equation. Now, we're not going to have to use that equation in this lesson, but there is just this idea from it that you should be aware of, which is that when the pH of a solution is exactly equal to the pKa of an acid, then exactly half of that functional group is going to be ionized, okay? So, what that means is that if your pH and your pKa are exactly equal to the same amount, that means that that group, that functional group will exist as a 50% neutral molecule and as a 50% charged molecule, maybe positive, maybe negative. It depends on the situation. That's neutral and charged. I'm sorry, it's a little bit off the screen. So what that means is that when they're at equilibrium like that, the neutral and charged forms are even with each other. But once you make the pH a little bit lower than the pKa, what's going to happen? Well, now, you're going to get a more protonated form that predominates because now there's going to be more acid around, so you're going to get more charges, more positives. And if the pH is higher than the pKa, then it's going to be more basic in the solution, so the deprotonated form is going to predominate, okay?

So I want to show you guys an example of what this could look like because it turns out that if you look at the pKa table for phenylalanine, the pKa1, or the pKa of the carboxylic acid group, it's not actually 2; it's close to 2, it's 1.83. You actually need to look that up, okay? You can't just say 2. The exact pKa2, or the pKa of the nitrogen, is not 9; it's 9.13, very similar but just a little bit higher. So, what that means is that at physiological pH of 7.4, like we already discussed, it's going to exist as a zwitterion. It's going to exist where the oxygen gives up its protons to the nitrogen so that the oxygen is negative, and so that the nitrogen is positive. And actually, this is going to be true for any pH all the way from 1.83 all the way to 9.13. So basically, for like 8 numbers of pH, it's going to look like this because in all these situations, the zwitterionic form is the most stable because in all these situations, the pH hasn't become a greater number than the pKa or a lesser number than the pKa.

But what starts to happen once you reduce the pH of the solution below 1.83? Well, when you think of it conceptually, if the pH is 1.83, is that acidic or basic? Super acidic, right? So, it means there's protons everywhere. So this negative charge has been holding on. It's been negative for a while. But what happens if I start slamming it with protons? Like I keep making it more and more acidic, right? What if I drop it to 1.83? What if I drop it to 1.5? What if I drop it to 1? What if I drop it to 0.5? You get what I'm saying? You keep throwing protons at that thing; eventually, it's going to get protonated. Eventually, at some point, if I add enough protons to it, I'm going to get a proton there. That's what 1.83 is. Any pH below 1.83, you add the proton. So that means that at pH 1.83 or below, the pH is less than the pKa1. pKa1 is 1.83, and that means that the protonated form predominates.

Now, the same thing with the nitrogen, so what happens if I start raising the pH of the solution all the way to like 10, all the way to 11, right? So this thing is getting super basic. There's hydroxide molecules everywhere. And at some point, one of these H? So, how do I know when the hydroxide is going to take away that H? You look at the pKa2 value. The pKa2 value is 9.13. So that means that any pH that is higher than 9.13, there's going to be so much base around that that nitrogen won't be able to hold on to that proton and eventually, it's going to lose that proton. Now, it doesn't become negatively charged, that's a different idea. But now, it used to be positive and now it goes into the neutral. And the deprotonated form, in this sense, doesn't mean that it always has a negative. And protonated doesn't always mean that it has a positive because actually, my protonated version didn't have a positive either. It just depends on the molecule you're starting with. But deprotonated means that I lost a proton, and protonated meant that I gained a proton. Does that make sense? Cool. So this is the way that we're going to be thinking about predominant forms. We're going to have to compare pKas using the sheet and then we're going to use the simple formula to figure out what form it should be in. So that's it for this video. Let's move on to the next one.

The 7 Ionizable Amino Acids

So you think you're getting the hang of this. This is kind of making sense. And now I'm going to throw a wrench into the whole thing. Because it turns out that there's an extra complication which is that 7 of the 20 amino acids that you learned actually also have ionizable side chains. What that means is that when you are planning out the zwitterionic form, if it has positives, negatives, etcetera, you need to also look at the side chain and you have to figure out if the side chain is going to be protonated or deprotonated, etcetera. On top of that, some professors, some universities make you memorize these 7 pKa's. So I'm going to give them to you and I don't really have the best memory tools for them. It's going to be up to you to figure out if you need to memorize these or if they're going to be given to you. Most of the time, 90% of the time, they're given to you just like the others, so you don't have to worry. But if you happen to have a professor that wants you to memorize them, then you're going to need to figure out a way to memorize them. Okay? So let's go ahead and move to the next page. I'm going to move to basically back to the amino acid breakdown page. Okay? So this is back to the amino acid breakdown and now we're going to be filling in these boxes over here and the 7 ionizable pKa's or side chains are the ones that have white boxes. So we're going to be filling in numbers here. Okay? Cool.

Another disclaimer. It turns out that not only does not everyone have to memorize them, some people do, some people don't, but there are also differing values depending on your professor, depending on what website you look at. Some will say 8.3 and some will say 8.4. It's like really stupid. So I personally don't think it's worth memorizing unless your professor makes you because no one really fully agrees. I'm going to go ahead and give you the values that I found were the most popular ones. But if your professor or textbook requires you to memorize a slightly different one, use that one. Okay? So cysteine, 8.18. I'm not going to give you ways to memorize this. I'm just going to write them down and you figure that out if you need to. Tyrosine, 10.07. Aspartic acid, 3.65. Glutamic acid, 4.25. By the way, that kind of makes sense. Right? That aspartic would be more acidic than glutamic because it's closer to the nitrogen, so it's going to have a stronger inductive effect and this one's further away. Kinda makes sense. Lysine, 10.53. Histidine is exactly 6. That one seems to be pretty universally agreed on. It's almost always 6. And then arginine, 12.48. Okay? So just I'm not going to help you memorize these. You probably don't even need to, but just have them written down so we can use them in the following problems. We're going to referring to this sheet a lot because we're going to need to get all our pKa values from here. Okay? Now, I happen to know that the next example is with Lysine. So let's get these Let's write down these numbers now so that we can use them on the next page. And I'm actually going to write them down myself so that I can use them on the next page. So lysine, the carboxylic acid is 2.18. The amine is 8.95. As you can see, again, they're close to 2.9, but they're a little bit off. And then the lysine side chain is 10.53. So now let's go over to the example, figure out what the predominant form of lysine looks like and what's the net charge at a pH of 8.5. Cool? So actually, let's just do this together. Okay? Actually, okay. No. I'll let you guys try it first, and then in the next video, I will solve it for you.

Predicting Predominant Form of Lysine

So let's write in our pKa's. This one, pKa 1, was 2.18. This one was 8.95. And then finally, this one was 10.53. Also, notice that I didn't include any hydrogens because the hydrogens are going to depend on the charge. So let's just go pKa by pKa, okay? So the first one. So remember, the pH is 8.5. So for pKa 1, is pH less than or greater than 2.18? It's greater than, right? So pH is greater than pKa 1. Does that make sense? And I'm just gonna write in the numbers just so you guys can see. pH is 8.5. pKa 1 is 2.18. Which one is bigger? pH, right? So what does that mean? Well, when the pH is greater, that means deprotonated. Right? We have to look at our rules. Deprotonated predominates, right, because it's basic. So I'm gonna put here deprotonated. And for this, deprotonated means it needs a negative charge. Cool. So we figured out the first charge already. Awesome. So let's look at the next one. So is the pH greater than or less than pKa 2? It's actually less than, right? Because pKa 2 is 8.95. So in this case, pH is less than pKa 2. And once again, just to make it really clear, what I'm saying is that 8.5 is a lower number than 8.95, correct? So since it's lower, that means that it's more acidic. That means protonated predominates. Let me avoid writing over the lysine. Okay? So that means that the protonated form of this amine is NH₃⁺. Right? So actually, we've got our zwitterion. At this pH of 8.5, we've got our zwitterion. By the way, I could have already known that because 8.5 is in between the two pKa's. Remember we said any pH that's between the two pKa's will form the zwitterion. So 8.5 makes sense that it does. Then finally, 10.53. So pH is less than pKa 3. And just to be super clear, that means that 8.5 is less than the number 10.53, right? So since it's less, that means it's more acidic which means that the protonated form should predominate. And the protonated form of the amine is NH₃. So that means that this should also be NH₃⁺. Cool. So now we figured out the predominant form at this specific pH. What is the net charge? Well, the net charge is just summing all the charges. So I have two positives and one negative. So that means that the net charge is equal to one positive. Okay? Or you could just put a bracket around and just say that it's positive. Okay? Cool. So now you guys get the fact that predominant forms aren't just with the backbone. You also may have to include the side chain and you may need to memorize your pKa's for the seven ionizable amino acids if your professor wants you to. But for right now, we'll just be looking at the reference sheet every time. Okay. So let's go ahead and move on to the next video.

Predict the predominant form and net charge of tyrosine (Y) at pH 10. What is the net charge? 

Predict the predominant form and net charge of tyrosine at a pH of 10 and then predict the net charge. So guys, in this problem, what we're going to have to do is draw the structure of tyrosine, look up the values of the pKa's of the carboxylic acid and the amine. And then probably look up the value of the ionizable side chain or potentially recall it if our professor wants us to memorize it, okay? I went ahead and I already wrote out the pKas for you so that we don't have to go back and forth between pages. Okay? But if I hadn't written that, you would just flip back to the breakdown sheet and write down those pKa's. Okay? So, what does tyrosine look like? Let's not draw Hs. It just looks like this. And then we've got our benzene ring because it looks like a tire, and then o. K? I'm not drawing H's because I don't know how many there's going to be yet. Cool. So then remember that we always refer to this as my pKa 1, my pKa 2, and my pKa 3, which are the same ones that I have written here, just so you know. K123. Great. And my pH is 10. So let's go right at it. For pKa 1, is the pH greater than or less than pKa 1? Much greater. The pH is greater than pKa 1. So if it's greater, that means it's more basic. So that means deprotonated. So that means that I should have a negative charge here. Okay. pKa 2. Is my pH greater than or less than pKa 2? It's greater than, right, because the pH is 10 and pKa 2 is 9.11. So it's greater. So that means that it should exist in the more basic deprotonated form as well. Now for an amine, deprotonated doesn't mean negative charge. It means that I draw it with an NH2, and it's neutral. Right? Because it used to have a positive. So deprotonating means that I took one of the protons away. So in this case, it's actually just going to be a neutral NH2. K? Finally, we're going to look at the last one. Is the pH greater than or less than pKa 3? So it is less than. It's a little bit less than. Once again, because my pH is 10 and my pKa 3 is 10.07. So, since this is slightly more acidic, it's going to be protonated. And that means the protonated form of an alcohol is just an alcohol. The deprotonated form would have been like an O negative. So this is just going to have its H. But if it had been just a little bit higher than it would have been a negative charge. Does that make sense? Cool. So now we've got everything down. We just need to figure out the net charge. The net charge is negative, neutral, neutral. It's just a negative charge. So, the net charge is equal to -1, which can also just be represented as putting brackets around it and putting a negative. And that would be your net charge. Alright. So we're done. Let's move on to the next one.

Determine the net charge of the dipeptide R-C at pH 4.3. (Hint:Peptide bonds do not count)

Determine the net charge of the dipeptide RC at pH 4.3. Hint: peptide bonds do not count. So this is really a little bit harder than I think any question you might get on your exam or in your homework. But it's great practice, so let's just try to do it anyway. And the thing is you can do this. Now, remember what this is going to involve is drawing my dipeptide from scratch and then looking up the pKa values for both arginine and cysteine. But I've already gone ahead to save time and drawn out pKa values for you. But if I had not done this, I would expect you to go to the amino acid breakdown sheet and get them from there. So let's go ahead and draw out our backbone first. So our backbone is going to look like this. Remember, I'm not going to draw hydrogen. So I'm going to draw nitrogen, nitrogen, O. Yeah. That looks good. And I'm also going to draw my R groups. So my R groups, this one's down and dashed. This one's up and wedged. Also, keep in mind, it says peptide bonds do not count. So that means that in no case do I have to figure out the pKa of this nitrogen. That's a peptide bond, so this one just gets a hydrogen just like always, okay? The only things I really need to worry about are the pKa's of I'm going to show you and I'm going to highlight the pKa's of these guys. This one, this one, this one, and this one. Does that make sense? Because peptide bonds in the middle, they don't count. Those are not ionized. Okay? So we're just looking for these 4 pKa's. Alright. So yeah, why don't we start in well, actually, you have to draw out our side chains, right? So arginine looks like this. Remember, arginine, step on it, hurts your foot. It's going to be pointy. So that's carbon, carbon, carbon, 1, 2, 3. And then nitrogen, nitrogen, nitrogen double bond. Now these 2 are not even impacted. The only nitrogen I care about is the terminal 1, okay? So remember when I said that you care about this pKa? Let's go ahead and erase that and move it over here because because of this side chain, this is the pKa I care about, Okay? That is just so you know, this is going to be pKa 3. And I'll erase these so that they're different colors. Okay? This nitrogen here is pKa3 of arginine. Cool? Actually, let's do that. Let's let's color code each of these even though you might not have the same markers as I do, but it will help for this lesson because there's a lot going on. What is pKa2 going to be for arginine? Or what is the nitrogen gonna be? Sorry. I gave it away. This nitrogen, pKa is going to be this 1, pKa2. K? How did I know that? Well, because remember, pKa 2 always has to do with the nitrogen. Right? It doesn't have to do with it has the oxygen would be pKa1. So I'd be saying, well, Johnny, then where how do I even use pKa 1 on the first one? You don't because the first one doesn't have an O. Right? It doesn't have an oxygen. So actually, it turns out that pKa 1 does not even get used in this question because there's no oxygen. Makes sense? Cool. Let's move on to the next one. So for the next one, I have to figure out the pKa of this guy, but I have to draw it first. So what does that functional group look like? Well, that's cysteine. So she's telling everyone to shut up in church. It's an S. Now I'm not going to draw the H because we don't know yet the protonated form. This is going to be this pKa. What pKa is that of cysteine? Is that pKa 1, 2, or 3? pKa 3, right? Because the side chain is always the third one. So it's going to be this one right here. Cool. Awesome. And then finally, we need to get one more pKa for this Is that going to be of cysteine? Is that going to be pKa 1 or pKa 2? Well, since it's oxygen, that's always pKa 1. That's the lowest one, the one that's close to 2. So in cysteine, we're actually not going to use pKa 2. And you say, Johnny, why? Why would you not use pKa 2? Because this nitrogen is in the peptide bond. And remember that peptide bonds don't count. You don't use them. So this is one of the differences between doing a question of just an amino acid versus of a peptide. If you do it of just an amino acid, you're using every pKa value possible. But once you have a peptide, you have to pick only the pKas that make sense. Okay? Because some of them are not going to exist because some of them are going to be peptide bonds. Some of them are going to be functional groups that are present. Does that make sense? So really, we're only comparing these 4 pKas, not 6 pKas. So let's go ahead and we'll just we'll just go in order starting with arginine pKa 2. Okay? So is the pH of 4.3 smaller or greater than arginine pKa 2? It's smaller, right? 4.3 versus 9.04. So I'm going to put here pH is smaller than arginine pKa 2. Does that make sense? And I'm just going to put the numbers just to make it super clear. 4.3 is a smaller number than 9.04. That means that the acidic form predominates which means that it is protonated. So let's fill that in. That means that this nitrogen here should actually be NH3+. I'll keep it yellow. Cool, right? So we know we have one positive. Let's go on to the next one. So pH is smaller or greater than arginine pKa 3. It's smaller than arginine pKa 3 because the number 4.3 is smaller than the number 12.48. Right? Way smaller. So since it's more acidic, that means the protonated form will predominate, which means that for this nitrogen group, it is also going to be protonated. Cool? So you have 2 down. 2 to go. So, let's go in order of the pKas in order like this. So now, I'm gonna do is the pH greater than or less than cysteine pKa 1? So it's actually greater than cysteine pKa 1. And that's because the pKa of 4 point the pH of 4.3 is a greater number than 1.96, right? So since it's more basic, that means the deprotonated form is gonna win. And that means that this is going to get a negative charge. And I'm running out of room, so I'm gonna do the last one up here. PH is greater than greater or equal I'm sorry, greater or smaller than cysteine pKa 3? It's smaller than cysteine pKa 3 because the number 4.3 is smaller than the number 8.18. Right? So that means it's more acidic. There's more protons around. That means that the protonated form will predominate. And the protonated form happens to be SH. Just like alcohol, protonated is SH. Deprotonated is S minus. It wouldn't be SH2+. Don't that's totally different. Okay? So we're done, guys. We drew our dipeptide perfectly by the way. Great job. And now, we figured out our the predominant forms. Now, we just need to do the net charge. And the net charge would be equal to positive, positive, neutral, negative. So it's 2 positives, one negative. It's just positive. So net charge, I would just put a bracket around this thing and I could say that it's positive and that is the end of this question. So great job. Wonderfully done. Let's move on to the next video.

Here’s what students ask on this topic:

What are zwitterions and how do amino acids exist as zwitterions at physiological pH?

Zwitterions are molecules that have both positive and negative charges but are overall electrically neutral. At physiological pH (around 7.4), amino acids exist as zwitterions. This occurs because the carboxyl group (–COOH) loses a proton (H⁺), becoming negatively charged (–COO⁻), while the amino group (–NH₂) gains a proton, becoming positively charged (–NH₃⁺). The result is a molecule with separated charges that balance out to a net charge of zero. This form is more stable at physiological pH due to the relative pKa values of the carboxyl and amino groups.

How do the pKa values of amino acids influence their charge at different pH levels?

The pKa values of amino acids determine the pH at which their functional groups (carboxyl and amino groups) gain or lose protons. When the pH is below the pKa of the carboxyl group (around 2), the carboxyl group remains protonated (–COOH). When the pH is above the pKa of the amino group (around 9), the amino group loses a proton (–NH₂). At intermediate pH levels, such as physiological pH (7.4), the carboxyl group is deprotonated (–COO⁻) and the amino group is protonated (–NH₃⁺), resulting in a zwitterion.

What is the Henderson-Hasselbalch equation and how is it used to determine the charge of amino acids?

The Henderson-Hasselbalch equation is used to relate the pH of a solution to the pKa of an acid and the ratio of the concentrations of its protonated and deprotonated forms. The equation is:

${\mathrm{pH}}_{}={\mathrm{pKa}}_{}+\frac{\log }{\frac{\mathrm{[A^-]}}{\mathrm{[HA]}}}$

In the context of amino acids, it helps determine whether the amino acid will be in a protonated or deprotonated state at a given pH. When pH = pKa, the concentrations of the protonated and deprotonated forms are equal.

Which amino acids have ionizable side chains and what are their pKa values?

Seven amino acids have ionizable side chains: cysteine (pKa ≈ 8.18), tyrosine (pKa ≈ 10.07), aspartic acid (pKa ≈ 3.65), glutamic acid (pKa ≈ 4.25), lysine (pKa ≈ 10.53), histidine (pKa ≈ 6.00), and arginine (pKa ≈ 12.48). These side chains can gain or lose protons depending on the pH, affecting the overall charge of the amino acid. The exact pKa values can vary slightly depending on the source, so it is important to refer to the specific values provided by your instructor or textbook.

How does the amphoteric nature of amino acids affect their behavior in different pH environments?

Amino acids are amphoteric, meaning they can act as both acids and bases. This dual behavior is due to the presence of both an acidic carboxyl group (–COOH) and a basic amino group (–NH₂). In acidic environments (low pH), amino acids tend to gain protons, becoming positively charged. In basic environments (high pH), they lose protons, becoming negatively charged. At physiological pH (around 7.4), they exist as zwitterions, with both positive and negative charges that balance out to a net charge of zero.